Use of hederagenin 3-0 alpha-l-rhamnopyranosyl(1-2)-(beta-d-glucopyranosyl(1-4)-alpha-l-arabinopyranoside or an extract from pulsatillae radix containing the same as a therapeutic agent for solid tumors

ABSTRACT

This invention relates to a use of hederagenin 3-O-α-L-rhamnopyranosyl((1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside or a Pulsatillae radix extract containing the same as a therapeutic agent for solid tumors. Also, this invention relates to a method of preparing a therapeutic agent for solid tumors by using hederagenin 3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside, or a method of treating a subject suffering from solid tumors, comprising administering to the subject a therapeutically effective dose of hederagenin 3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside.

This application is a divisional of U.S. patent application Ser. No.11/107,665, filed on Apr. 15, 2005, which in turn was acontinuation-in-part of U.S. patent application Ser. No. 10/620,709filed on Jul. 15, 2003.

TECHNICAL FIELD

This invention relates to a use of hederagenin 3-O-α-L-rhamnopyranosyl((1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside as represented bythe following formula (I):

or a water-soluble fraction of Pulsatillae radix containing the same asa therapeutic agent for solid tumors.

Also, this invention relates to a method of preparing a therapeuticagent for solid tumors by using hederagenin3-0-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside,and to a method of treating a subject suffering from solid tumors,comprising administering to the subject a therapeutically effective doseof hederagenin3-0-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside.

BACKGROUND ART

Pulsatillae radix is a dried root of Pulsatilla species belonging to theRanunculaceae family (Ki Hwan Bae, Korean Medicinal Herbs, 1999).According to the Chinese medicine, Pulsatillae radix is known to haveeffects of removing heat from the blood and detoxifying. It has alsobeen used as anti-inflammatory, astringent, hemostatic and antidiarrheaagents, and for the treatment of hematochezia, malaria, nosebleed, andbleeding from tooth. Its flower is called as Pulsatillae Flos, and usedfor the treatment of malaria, or smallpox. Its leaf is called asPulsatillae Folium, and used for treatment of waist pain, edema, orheart pain. In addition, decoction of Pulsatillae radix was reported tohave an antibacterial effect against amoebic dysentery, and a pesticidaleffect against Trichomonas.

Pulsatillae radix contains about 9% of saponins, and such ingredients asprotoanemonin, anemonin, ranunculin, hederagenin, betulinic acid, andoleanolic acid derivatives and their glycosides have been isolatedtherefrom by now as represented by the following formula (II):

The above ingredients have not yet been extensively studied for theirpharmacological effects, but protoanemonin was reported to havemitotoxicity (Vonderbank, F., Pharmazie 5, 210, 1950). Li, et al. (Li,R. Z., et al., Yao Hsueh Hsueh Pao. 28, 326 31, 1993) also reported thatranunculin has cytotoxicity against KB cells, by inhibition of DNApolymerase.

Hederagenin3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosidewas isolated from Pulsatilla cerna and P. koreana by Shimizu, et al.(Chem. Pharm. Bull., 26, 1666, 1978); from P. chinensis by Yoshihiro, etal. (J. Nat. Pro., 62, 1279, 1999); and from Serjania salzmannianaSchlecht by Ekabo, et al. (J. Nat. Prod., 59, 431, 1996). Kang, et al.(Arch. Pharm. Res., 12(1), 42-47, 1989) also isolated it from P.koreana, and reconfirmed its structure. Yoshihiro, et al. reported thathederagenin and oleanolic acid derivatives showed cytotoxicity againstHL-60 human leukemia cells in the above article. They reported thathederagenin3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosideisolated from Chinese Pulsatillae radix (Pulsatilla chinensis) had weekcytotoxicity, i.e. 3.8 μg/ml of ED₅₀, against HL-60 cells. However, mostof saponins and many kinds of natural products commonly show such levelof cytotoxicity, and thus, the above compound cannot be said to haveantitumor activity based thereon. Therefore, it has never been knownthat hederagenin3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosidehas antitumor activity, particularly, against solid tumors.

DISCLOSURE OF THE INVENTION

The present inventors isolated deoxypodophyllotoxin from medicinal herbsincluding Anthriscus sylvestris Hoffman, Pulsatillae radix, etc., andfound that this substance inhibited the growth of solid tumor cells byinhibiting angiogenesis, and obtained a Korean patent (Korean PatentNumber 315,200) for the same. The present inventors carried outextensive studies to develop an antitumor agent from medicinal herbs. Asa result, they obtained a fraction which is poorly soluble in an organicsolvent, but is readily soluble in water from Pulsatillae radix, andisolated an antitumor compound from the fraction, and so completed thepresent invention.

Accordingly, the purpose of the present invention is to provide atherapeutic agent for solid tumors comprising an antitumor compoundisolated from Pulsatillae radix (hederagenin3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside)or a water-soluble fraction of Pulsatillae radix containing the same asan active ingredient, a method of preparing a therapeutic agent forsolid tumors by using hederagenin3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside,or a method of treating a subject suffering from solid tumors,comprising administering to the subject a therapeutically effective doseof hederagenin3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside.

One aspect of the present invention provides a therapeutic agent forsolid tumors comprising hederagenin3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosideas an active ingredient.

Another aspect of the present invention provides a therapeutic agent forsolid tumors comprising hederagenin3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosideas an active ingredient, wherein said hederagenin3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosideis contained in a water-soluble fraction of Pulsatillae radix obtainedby extracting Pulsatillae radix with an aqueous solution of ethanol, andforming precipitates by adding acetone thereto.

Another aspect of the present invention provides a method of preparing atherapeutic agent for solid tumors by using hederagenin3-0-α-L-rhamnopyranosyl(1→2)-[13-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside.

Another aspect of the present invention provides a method of preparing atherapeutic agent for solid tumors by using hederagenin3-0-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside,wherein said hederagenin3-0-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosideis contained in a water-soluble fraction of Pulsatillae radix obtainedby extracting Pulsatillae radix with an aqueous solution of ethanol, andforming precipitates by adding acetone thereto.

Preferably, the water-soluble fraction of Pulsatillae radix is afraction having R_(f) in the range of 0.48 to 0.50, and developing redcolor and then blue color, upon spraying sulfuric acid followed byheating, wherein the water-soluble fraction of Pulsatillae radix isprepared by extracting Pulsatillae radix with an aqueous solution ofethanol, forming precipitates by adding acetone thereto to obtain awater-soluble fraction, and passing the fraction through a Sephadex LH20column.

Another aspect of the present invention provides a method of treating asubject suffering from solid tumors, comprising administering to thesubject a therapeutically effective dose of hederagenin3-0-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside.

Another aspect of the present invention provides a method of treating asubject suffering from solid tumors, comprising administering to thesubject a therapeutically effective dose of hederagenin3-0-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside,wherein said hederagenin3-0-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosideis contained in a water-soluble fraction of Pulsatillae radix obtainedby extracting Pulsatillae radix with an aqueous solution of ethanol, andforming precipitates by adding acetone thereto.

Preferably, the water-soluble fraction of Pulsatillae radix is afraction having R_(f) in the range of 0.48 to 0.50 and developing redcolor and then blue color, upon spraying sulfuric acid followed byheating, wherein the water-soluble fraction of Pulsatillae radix isprepared by extracting Pulsatillae radix with an aqueous solution ofethanol, forming precipitates by adding acetone thereto to obtain awater-soluble fraction, and passing the fraction through a Sephadex LH20column.

“Solid tumors,” as used herein, refer to any mass tumor except bloodcancers, a representative example of which is lung tumor.

In the present invention, the Pulsatillae radix extract containinghederagenin3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosidecan be obtained by extracting Pulsatillae radix with an aqueous solutionof ethanol, and forming precipitates by adding acetone thereto to obtaina water-soluble fraction (WT). Or, it can be obtained by extractingPulsatillae radix with the aqueous solution of ethanol, formingprecipitates by adding acetone thereto to obtain the water-solublefraction, and passing the fraction through Sephadex LH20 column toobtain a fraction (SPX3) having R_(f) of 0.48˜0.5, and developing redcolor, and then, blue color upon spraying sulfuric acid followed byheating.

Hereinafter, the present invention will be explained in detail.

According to the present invention, Pulsatillae radix extract isextracted with 50% ethanol to obtain a fraction WT poorly soluble inacetone, and the fraction is further purified on Sephadex LH20 to obtainfraction SPX3, and from the SPX3 fraction, pure SB365 is finallyobtained. This compound is hederagenin3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside,and exhibits higher antitumor activity against solid tumors formed withmouse lung tumor cells, LLC (Lewis Lung Carcinoma) cells, or human lungtumor cells, NCI-H23 cells, than a clinical drug, adriamycin.

The water-soluble fraction of Pulsatillae radix according to the methodof the present invention is extracted with 50% ethanol, and acetone isadded to the ethanol extract to remove acetone-dissolved part, and sothe resulting water-soluble fraction of Pulsatillae radix comprises highconcentration of hederagenin3-0-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside.

According to an experiment for the content of hederagenin3-0-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside,the water-soluble fraction of Pulsatillae radix according to the methodof the present invention contains an average of 1.20 mg of hederagenin3-0-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosideper g. Therefore, the water-soluble fraction of Pulsatillae radixaccording to the method of the present invention provides superiorantitumor activity against solid tumors.

In particular, the present process for preparing an antitumor fractionfrom Pulsatillae radix and isolating an antitumor substance therefrom isas follows.

(1) Preparation of Antitumor Fraction Wt from Pulsatillae Radix

Pulsatillae radix powder was extracted with 50% aqueous solution ofethanol, and dried under reduced pressure. To the obtained driedmaterial was added acetone at 5 to 10-fold amount. The mixture wasshaken, centrifuged at 3,000 rpm, and the supernatant was decantedtherefrom to obtain an insoluble part. The above process was repeatedtwice. The remaining insoluble part was readily soluble in water, and sodesignated as “fraction WT.” This fraction showed relatively highantitumor activity against BDF1 mice transplanted with LLC cells andnude mice transplanted with NCI-H23 cells.

(2) Preparation of Fraction Spx3 from Fraction Wt

A given amount of fraction WT is dissolved in a given amount of aqueoussolution of methanol at various concentrations, and then fractionated onSephadex LH20 column stabilized with the same solvent. In this case, thebest isolation is achieved with employing 80% aqueous solution ofmethanol, and the suitable size of the filled column was 60×4 cm for 500mg of fraction WT. As a result, fraction SPX1 (test tube numbers 26-66),SPX2 (test tube numbers 66-91), SPX3 (test tube numbers 91-111), andSPX4 (test tube numbers 111-138) were obtained. When spraying sulfuricacid onto the fractions developed on a silica gel plate and heating theplate, the fraction SPX3 developed red color at first, and blue colorwith the lapse of time, and contains a spotted compound having R_(f) of0.48 to 0.50 as a main ingredient. It was shown to have high antitumoractivity on BDF1 mice transplanted with LLC cells and nude micetransplanted with NCI-H23 cells.

(3) Isolation of SB365 from Fraction SPX3

To isolate an antitumor substance from the fraction SPX3 showingantitumor activity, HPLC was carried out to obtain pure compound, SB365.To identify the structure of SB365, Lieberman-Burchard reaction, IR,¹H-NMR, ¹³C-NMR, and ethanol/sulfuric acid hydrolysis were carried out.As a result, SB365 was confirmed as hederagenin3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside,which is a saponin ingredient that had already been isolated fromPulsatillae radix.

The Pulsatillae radix extracts according to the present invention orpure SB365 compound isolated therefrom have a weak cytotoxicity againstsolid tumor cells, but unexpectedly showed excellent antitumor activityin animal experiments. Thus, they can improve problems caused byprevious antitumor agents on clinical use, e.g. reducing immuneresponses due to decreasing blood cells. It is also anticipated thatthey show low toxicity on rapidly dividing cells like hematopoieticcells, etc.

The Pulsatillae radix fractions and SB365 according to the presentinvention may be combined with pharmaceutically acceptable carriers thatare conventionally used, and manufactured into various formulations thatare conventional in the pharmaceutical field, for example, orallyadministrable formulations like solutions, suspensions, etc.; injectableformulations like injectable solution or suspension, ready-to-useinjectable dry powder, etc.; and topically administrable formulationslike ointments, creams, and solutions. Particularly, the activeingredient of the present invention is soluble in water, and may bedissolved in various solutions such as physiological saline, Ringer'ssolution, and nutrient solution, etc. Such pharmaceutical formulationsmay be intravenously, subcutaneously, intraperitoneally, or topicallyadministered.

A recommended dosage of the active ingredient of the present inventionto human beings is 3.5˜8.0 mg/kg body weight in case of SB365, 20˜40mg/kg body weight in case of fraction SPX3, or 200˜300 mg/kg in case offraction WT. The optimal dosage is 6.5 mg/kg body weight in case ofSB365, 25 mg/kg body weight in case of fraction SPX3, or 250 mg/kg bodyweight in case of fraction WT. However, such dosage may be appropriatelyadjusted depending on age, body weight, health, severity of disease ofpatients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows silica gel TLC patterns of fraction WT;

FIG. 2 shows silica gel TLC patterns of fraction SPX3 purified on aSephadex LH20 column;

FIG. 3 is an HPLC chromatogram of fraction SPX3; and,

FIG. 4 is an HPLC chromatogram of SB365.

BEST MODE FOR CARRYING OUT THE INVENTION

This invention will be better understood from the following examples.One skilled in the art will readily appreciate the specific materialsand results described are merely illustrative of, and are not intendedto, nor should be intended to, limit the invention as described morefully in the claims, which follow thereafter.

Example 1 Preparation of Fraction Wt

Pulsatillae radix powder of 50 g was extracted three times with 500 mlof 50% aqueous solution of ethanol, and the extract was dried underreduced pressure to obtain 22 g of dried materials. To this driedmaterials was added 300 ml of acetone, and the mixture was shaken andcentrifuged at 3,000 rpm. The supernatant was removed therefrom to givea precipitate. For this precipitate, the acetone treatment was repeatedtwice. The acetone layer was discarded, and an insoluble part was driedto obtain 17.8 g of dried materials (fraction WT). The obtained fractionWT was subjected to silica gel TLC (developing solvent: butanol:aceticacid:water in the ratio of 4:1:1, color reaction: sulfuric acid-sprayingfollowed by heating). The result is shown in FIG. 1. In FIG. 1, a bluespot having the R_(f) in the range of 0.48 to 0.50 corresponds to theactive ingredient of the present invention as described below. As shownin Experimental Example 1 below, fraction WT showed relatively highantitumor activity (inhibition rate of tumor growth: 57%) on BDF1 micetransplanted with LLC cells.

Example 2 Preparation of Fractions SPX

Fraction WT of 560 mg was further fractionated on Sephadex LH20 column(200 g, 60×4 cm) using a mixed solution of methanol and water (80:20)with the flow rate of 1 ml/min, and the fraction volume of 0.5 ml/tube.These fractions were spotted on a silica gel thin layer in order, anddeveloped to obtain factions (developing solvent: butanol:aceticacid:water in the ratio of 4:1:1, color reaction: sulfuric acid-sprayingfollowed by heating). The result is shown in FIG. 2. In FIG. 2, SPX1(139 mg, 24.8%) was obtained by collecting test tube numbers 26 to 66,and consisted of 4 major spots, lower one of which developed yellowcolor upon reacting with sulfuric acid. SPX2 (344 mg, 61.4%) wasobtained by collecting test tube numbers 66 to 91, and consisted of 2major spots. SPX3 (61 mg, 10.9%) was obtained by collecting test tubenumbers 91 to 111, and developed red color at first, and then blue colorwith the lapse of time, upon spraying sulfuric acid followed by heating.Fraction SPX3 contained a spot having the R_(f) value in the range of0.48 to 0.50 as its major ingredient. SPX4 (15.7 mg, 2.8%) was obtainedby collecting test tube numbers 111 to 138. Fractions SPX3 and SPX4 hadrelatively high purity showing one spot on the thin layer.

As shown in Experimental Example 1 below, SPX3 exhibited 60% of theinhibition rate of tumor growth on 15 days from its administration. Bycontrast, SPX1, SPX2, and SPX4 did not exhibit any action, and so itcould be assumed that the substance developing blue color againstsulfuric acid was an ingredient with antitumor activity. This SPX3fraction may be used as an antitumor agent in itself.

Example 3 Isolation of SB365

In order to isolate a pure substance from fraction SPX3, HPLC wascarried out as follows.

Reverse-phased silica gel (RP-C₁₈, 250×10 mm, manufactured by Metachem)was used as the fixed phase, and a mixed solution of methanol and water(80:20) was used as the mobile phase. The detection wavelength was 210nm, and the flow rate was 1 ml/min. The result is shown in FIG. 3. Asshown in FIG. 3, SPX3 consisted of 3 major substances. From the obtainedfractionated amounts, peaks at R_(t) of 8.5 min and 10.4 min containedsmall amounts of ingredients, and a peak at R_(t) of 23.3 min containedthe major ingredient. Thus, it was assumed that the latter would haveantitumor activity. As described above, the substance with R_(t) of 23.3min that developed blue color with sulfuric acid and would be the activeingredient was collected to obtain SB365 of 2.8 mg from 31 mg of SPX3.The collected fraction at R_(t) of 23.3 min was dried, and was subjectedto HPLC under the condition as described above to measure its purity.The result is shown in FIG. 4. From FIG. 4, it was confirmed that SB365was a pure substance. The obtained SB365 was directly used for thestructural identification and antitumor activity test below.

As shown in Experimental Examples 1 and 2 below, SB365 exhibited 81% and82.1% of the inhibition rate of tumor growth on BDF1 mice transplantedwith LLC cells and nude mice transplanted with NCI-H23 cells,respectively, which could be said to be excellent antitumor activities.

Example 4 Structural Identification and Confirmation of the ActiveCompound Sb365

SB365 isolated in the above was the white amorphous form with m.p.239˜241° C. and [α]_(D)+23.6° (c, 0.2, MeOH), and was positive inLiebermann-Buchard reaction, and so confirmed as a glycoside. Inaddition, according to IR (cm⁻¹), peaks were observed at 3400 (br, —OH),2940 (br, C—H), 1695 (C═O), 1455, and 1040 (C═O). It was also assumed asa glycoside from the absorption peaks in the ranges of 1000-1100 and3000-3400.

In view of ¹H-NMR, it had NMR patterns typical of saponins. Six —CH₃groups were observed at 0.91, 0.92, 0.98, 1.00, 1.07, and 1.21 ppm, andanother —CH₃ group was observed as doublet at 1.64 ppm. It could be seenfrom this that the compound comprised one rhamnose group in its sugargroups. Anomeric protons were observed at 6.25 (br.), 5.11 (1H, J=7.80Hz), and 4.97 ppm (1H, J=6.66 Hz). Therefore, SB365 was confirmed as aglycoside having three sugar groups.

According to ¹³C-NMR, a hydroxymethyl group was observed at 65. 4 ppm(C-23), and three anomeric carbon signals was observed at 104.2 (C-1′),106.7 (C-1″′), and 101.7 ppm (C-1′). Two olefinic carbons were observedat 122.5 ppm (C-12) and 144.8 ppm (C-13), and one carboxy carbon wasobserved at 180.2 ppm (C-28). In general, about 4 Hz of glycosylationupfield shift is shown when sugar is bound at the 28 position (180.2ppm→176.2 ppm). In the present compound, the above phenomenon was notobserved, and so it was confirmed that the compound does not have asugar group in the 28 position.

Subsequently, the compound was hydrolyzed in ethanol/sulfuric acid toidentify its sugar groups and the structure of aglycone. SB365 wasconfirmed as hederagenin after comparing physicochemical data of thehydrolysis product, aglycone, ¹³C-NMR, and ¹H-NMR data. Further, thehydrolyzed sugars were confirmed as rhamnose, arabinose, and glucose bycomparative TLC.

On the basis of the above analysis results and data in publishedliteratures, SB365 was confirmed as hederagenin3-O-α-L-rhamnopyranosyl((1→2)-[β-D-glucopyranosyl(1→4)]-α-arabinopyranoside.

¹H-NMR and ¹³C-NMR data of SB365 are as shown in the following Table 1.

TABLE 1

Position ¹H (ppm) J (Hz) ¹³C (ppm) C-1  38.9 C-2 26.1 C-3 3.28 d 10.981.0 C-4 43.5 C-5 48.1 C-6 18.1 C-7 32.8 C-8 39.7 C-9 47.8 C-10 36.9C-11 23.9 C-12 5.45 s 122.5 C-13 144.8 C-14 42.1 C-15 28.3 C-16 23.8C-17 46.2 C-18 41.9 C-19 46.4 C-20 30.9 C-21 34.2 C-22 33.2 C-23 4.36,3.67 overlap 65.4 C-24 1.07 s 14.0 C-25 0.91 s 16.0 C-26 0.98 s 17.4C-27 1.21 s 26.3 C-28 — 180.2 C-29 0.92 s 32.8 C-30 1.00 s 23.7Arabinose C-1′ 4.97 d 6.66 104.2 C-2′ 80.4 C-3′ 75.4 C-4′ 76.2 C-5′ 63.9Rhamnose C-1″ 6.25 br 101.7 C-2″ 72.3 C-3″ 72.4 C-4″ 74.1 C-5″ 69.6 C-6″1.64 5.94 18.6 Glucose C-1′″ 5.11 d 7.80 106.7 C-2′″ 75.0 C-3′″ 78.5C-4′″ 71.2 C-5′″ 78.8 C-6′″ 62.5

Experimental Example 1 Antitumor Activity on BDF1 Mice Transplanted withLLC Cells

The mouse species used in this experiment was BDF1, and healthy malemice with the body weight of 18˜25 g were used. These animals weresupplied with water and foods ad libitum at a place of controlledtemperature in the range of 23˜24° C., and were bred with anantibiotic-free mouse feed. LLC cells were subcutaneously cultured inC57BL/6 mice for 14 days. A LLC cell-containing tissue was taken andthereto was added sterilized cold physiological saline water (5 ml/gtissue) to prepare a cell suspension. The cell suspension of 0.2 ml wassubcutaneously transplanted to the groin region of BDF1 mouse.

From 24 hours after transplantation, the above mice were divided intoseveral groups consisting of 5 mice. Then, samples, fractions WT and SPXfractions, and SB365, were dissolved in physiological saline, and wereinjected intraperitoneally at each concentration of 280 mg/kg (WT), 70mg/kg (SPX1), 171 mg/kg (SPX2), 30.5 mg/kg (SPX3), 8.1 mg/kg (SPX4), and6.4 mg/kg (SB365). To the negative control group was injected only thephysiological saline, and to the positive control group was injectedadriamycin (0.5 mg/kg). The injection was scheduled from 24 hours aftertumor transplantation to administer the samples successively once a dayfor 7 days, and stopped for one day, and then, was carried out for 6more consecutive days.

In order to evaluate toxicity of SB365 on mice, experimental mice wereweighed twice a week. Antitumor activity was calculated after measuringtumor volume of the control and test groups on 14th and 15th day aftersample administration as follows:

Tumor volume (mm³)=length (mm)×width (mm²)/2

Inhibition rate of tumor growth (%)=(C-T)×100/C

(C: average tumor volume in the control group, T: average tumor volumein the test group)

The result is shown in the following Table 2.

TABLE 2 Inhibition rate of tumor growth (IR, %) of Pulsatillae radixfractions and SB365 on BDF1 mice transplanted with LLC cells Fractionsor Number Inhibition rate of tumor growth (%) compounds of mice 14thday^(a)) 15th day^(a)) WT 5 56 55 SPX1 5 10 12 SPX2 5 25 30 SPX3 5 57 60SPX4 5 8 10 SB365 5 82 79 Adriamycin 5 60 64 ^(a))Days aftertransplantation of tumor cells

As shown in the above Table 2, fractions WT and SPX3 showed theinhibition rate of tumor growth of 55% and 60%, respectively, and SB365showed the inhibition rate of tumor growth of 79%, higher thanadriamycin of 64% on 15th day from transplantation of tumor cells.

Experimental Example 2 Antitumor Activity on Nude Mice Transplanted withNCI-H23 Cells

Female nude mice of the age of 5 weeks weighing 16˜25 g obtained fromHarlan Co. (USA) were used as experimental animals in this experiment.The mice were used after acclimation for 1 week in an aseptic animalroom. The animal room maintained the temperature of 22±2° C., thehumidity of 55±5%, and the light and darkness cycle of 12 hours, whichwas automatically controlled. Solid feed for experimental animals wasradiosterilized, and drinking water was sterilized in an autoclave. Theanimals were supplied with feed and drinking water ad libitum. A humantumor cell line provided by National Cancer Institute (NCI), USA, andpreserved in the Korean Research Institute of Bioscience andBiotechnology (KRIBB), Korea, was used. Lung tumor cells, NCI-H23 cells,among the human tumor cells, were transplanted to the nude mice. Thetumor cells of 3×10⁷ cells/ml were subcutaneously transplanted to themice at a volume of 0.3 ml/20 g body weight. The samples wereintraperitoneally injected to the mice every day for 13 days, that is,from 1 day to 14th day except 8th day after tumor cells transplantation.The size of tumor formed during the injection was measured in eachanimal, and any change in its body weight was also measured. On 16th dayafter tumor cells transplantation, the nude mice were sacrificed, andthe tumor was separated and weighed. To the positive control group wasintraperitoneally injected adiamycin of 0.5 mg/kg body weight on 1st,5th, 9th, and 14th day. The result is shown in the following Table 3.

TABLE 3 Inhibition rate of tumor growth (IR, %) of SB365 on nude micetransplanted with NCI-H23 cells Inhibition rate of tumor growth (%) onNCI-H23 Adriamycin SB365 SB365 SB365 Negative (0.5 (1.6 (3.2 (6.4 Groupcontrol mg/kg) mg/kg) mg/kg) mg/kg) 16th day^(a)) — 61.5 40.1 52.3 82.1^(a))Days after tumor cells transplantation

As shown in the above Table 3, SB365 of 6.4 mg/kg showed a highinhibition rate of tumor growth, 82.1%, on 16th day after tumor cellstransplantation.

Experimental Example 3 Cytotoxicity Test

Tumor cells A549, SK-MEL-2, and MCF-7 were obtained from the KRIBB, andused in this experiment. A culture medium was prepared by adding onepack of L-glutamine-containing RPMI1640 medium, 100 ml of fetal bovineserum (FBS) inactivated by heating at a water bath of 50° C. for 30minutes, 2 g of NaHCO₃, 100,000 units of penicillin, and 100 mg ofstreptomycin, to a sterilized distilled water for injection, adjustingthe pH of the mixture with 0.1 N HCl to a total volume of 1 l, anddisinfecting the mixture with filtration, and stored at 4° C. beforeuse. The cells were maintained by propagation once every three days, anda solution containing 0.5% trypsin and 2% EDTA in physiological bufferedsaline (PBS) was used to detach the cells from wells.

Cytotoxicity on tumor cells was measured according to Sulforhodamine-B(SRB) method developed by the NCI in 1989 to measure in vitro antitumoractivity of drugs.

Specifically, the cells were detached from wells with 0.5% trypsin-EDTAsolution and then, 3˜5×10⁴ cells/ml of cell suspension was prepared.Then, the cell suspension (180 μl/well) was added to 96-well plate, andthe plate was incubated in an incubator of 37° C., 5% CO₂ for 24 hours.

The sample was dissolved in dimethylsulfoxide (DMSO) and diluted withthe culture medium or tertiary distilled water to obtain requiredconcentrations for experiment, and serially diluted to a finalconcentration of DMSO of 0.2% or less. To each well of 96-well platewere added 20 μl of the serially diluted sample solutions, and then, theplate was incubated in an incubator of 37° C., 5% CO₂ for 48 hours. Atthe point of time to add the sample solution, T_(z) (Time zero) platewas collected. Medium was removed from T_(z) plate and from each plateafter completing the incubation, and to the plates was added 10%trichloroacetic acid (TCA) (50 μl/well). The resulting plates wereallowed to stand for 1 hour at 4° C. to immobilize the cells on thebottom of the plates. After completing the cell immobilization, theplates were washed 5˜6 times with water to completely remove theremaining TCA solution, and the resulting plates were dried at roomtemperature to contain no moisture.

To the completely dried plates was added 50 μl of a staining solutionwith 0.4% SRB in 1% acetic acid to stain the cells for 30 minutes. Then,the plates were washed 5˜6 times with 1% acetic acid solution tocompletely remove SRB unbound to the cells. The plates were dried atroom temperature. Thereto was added 100 μl of 10 mM Tris solution todissolve the dye. Then, OD (optical density) value was measured bymicroplate reader at a wavelength of 520 nm.

ED₅₀ value of the sample on tumor cells [50% effective dose (ng/ml): aconcentration at which tumor cell growth is inhibited by 50%] wascalculated as follows. T, value was defined as OD value at the time ofstarting the incubation after adding the sample, C (control) value as ODvalue of the well not treated with the sample, and T (test) value as ODvalue of the well treated with the sample. From the values T_(z), C, andT, cytotoxicity of the agent was measured by the following formula:

in the case of T _(z) ≧T, (T−T _(z))/(C−T _(z))×100

in the case of T _(z) <T, (T−T _(z))/T _(z)×100

From the values as calculated above, ED₅₀ value of the sample wasobtained by using data regression function of Lotus program.

As a result, ED₅₀ value of SB365 on human lung tumor cells, A549 cells,human melanoma cells, SK-MEL2, and human breast tumor cells, MCF7was >20 μg/ml, >10 μg/ml, and >10 μg/ml, respectively. Therefore, SB365had little cytotoxicity on solid tumor cells.

Formulation Example 1 Preparation of an Injectable Solution ContainingFraction WT

WT fraction of 250 mg obtained in Example 1 was dissolved in 10 ml ofphysiological saline to prepare an injectable solution.

Formulation Example 2 Preparation of an Injectable Dry Powder ContainingFraction SPX3

SPX 3 fraction of 25 mg obtained in Example 2 was dissolved in 10 ml ofRinger's solution, sterilized and then, freeze-dried to prepareready-to-use injectable dry powder. This powder would be re-constitutedwith distilled water for injection before use.

Formulation Example 3 Preparation of an Injectable Solution ContainingSB365

SB365 of 6.5 mg obtained in Example 3 was dissolved in 10 ml of Ringer'ssolution and sterilized to prepare an injectable solution.

INDUSTRIAL APPLICABILITY

Pulsatillae radix factions WT and SPX3, and SB365, hederagenin3-O-α-L-rhamnopyranosyl((1→2)-[β-D-glucopyranosyl(1→4)]-α-arabinopyranoside,isolated from the fractions according to the present invention, not onlyhave a high inhibition rate of tumor growth on solid tumor cells, butalso can be conveniently used by dissolving in various solutionsincluding physiological saline, Ringer's solution, or nutrient solutionbecause it is readily soluble in water, and has low cytotoxicity enoughto ameliorate side effects of previously developed anti-tumor agents.Therefore, it is anticipated to be very useful as a therapeutic agentfor solid tumors. In particular, the Pulsatillae radix extract accordingto the method of the present invention contains high concentration ofhederagenin3-O-α-L-rhamnopyranosyl((1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside,and so the present invention can provide superior antitumor activityagainst solid tumors.

1. A method of treating solid tumors in a subject in need thereof,comprising administering to the subject a therapeutically effective doseof hederagenin3-0-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranoside.2. The method according to claim 1, wherein said hederagenin3-0-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosideis contained in a precipitate obtained by extracting Pulsatillae radixwith an aqueous solution of ethanol to form a mixture, adding acetone toprecipitate the mixture and collecting the precipitate formed.
 3. Themethod according to claim 1, wherein said hederagenin3-0-α-L-rhamnopyranosyl(1→2)-β-D-glucopyranosyl(1→4)-α-L-arabinopyranosideis contained in a fraction obtained by extracting Pulsatillae radix withan aqueous solution of ethanol to form a mixture, adding acetone toprecipitate the mixture, collecting the resultant precipitate, dryingthe precipitate, reconstituting the precipitate, fractionating thereconstituted precipitate by gel filtration, subjecting the fractions tothin layer chromatography and collecting a fraction which has an Rfvalue of 0.48 to 0.50.
 4. The method according to claim 1, whichcomprises administering to the subject a therapeutically effective doseof hederagenin3-0-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosideby dissolving hederagenin3-0-α-L-rhamnopyranosyl(1→2)[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosidein a solution selected from the group consisting of physiologicalsaline, Ringer's solution, and nutrient solution. 5-11. (canceled) 12.The method according to claim 1, wherein the hederagenin3-O-α-L-rhamnopyranosyl((1→2)-[β-D-glucopyranosyl(1→4)]-α-arabinopyranosideis administered at a daily dose of 3.5 to 8 mg/kg body weight.
 13. Themethod according to claim 2, wherein the precipitate is administered ata daily dose of 200 to 300 mg/kg body weight.
 14. The method accordingto claim 3, wherein the fraction is administered at a daily dose of 20to 40 mg/kg body weight.